RT issues and efficiency of cDNA synthesis - qRT-PCR (Feb/15/2006 )

is there a way to measure the efficiency of the RT reaction and the quality of the first strand cDNA created?

I have seen lots of products for RNA extracted from mammalian cells. How about for bacterial RNA?

Also in a recent TaqMan Assay my -RT control had a Ct value of 32, the Ct in my NTC was 40. According to my standard curve the -RT control corresponded to 1.56E-3 ng of DNA, quite low compared to my samples which were at a Ct of ~18 and corresponded to 31 ng of DNA

As much as it will be impossible to remove all DNA contamination in any RNA extraction, at which level can it become an issue in qRT-PCR?What values are usually expected for a -RT in a qRT-PCR assay?ThanksR.

-raisinbread-

QUOTE (raisinbread @ Feb 15 2006, 08:06 PM)

Hello

is there a way to measure the efficiency of the RT reaction and the quality of the first strand cDNA created?

I have seen lots of products for RNA extracted from mammalian cells. How about for bacterial RNA?

Also in a recent TaqMan Assay my -RT control had a Ct value of 32, the Ct in my NTC was 40. According to my standard curve the -RT control corresponded to 1.56E-3 ng of DNA, quite low compared to my samples which were at a Ct of ~18 and corresponded to 31 ng of DNA

As much as it will be impossible to remove all DNA contamination in any RNA extraction, at which level can it become an issue in qRT-PCR?What values are usually expected for a -RT in a qRT-PCR assay?ThanksR.

Just to throw out some numbers.

I usually generate 4-6 micrograms of cDNA from 10 micrograms of starting total RNA using superscript or arrayscript M-MLV and nine micrograms of primer.

Extracting RNA from bacterial cells is trickier because usually some of the mRNA has short half-lives (15-30 mins) and efficient cell lysis is more challenging.

My guess from the Ct values you have provided is that you are not sufficiently lysing your cells for the RNA extraction. What bacteria are you extracting RNA from and what is your protocol?

As for DNA contamination, I would recommend DNase I treatment with something like Turbo DNA free from Ambion prior to reverse transcription.

You should expect your ct values to be below 30 for the rt treatment and above 32 for the no rt treatment with SYBR (at least).